Chromatographic assay systems employed as rapid assay devices are one of several means for detecting the presence of a given analyte in a biological sample. One advantage to these systems is that the execution of these assays does not use additional specialized equipment or trained personnel. Another advantage is the great variety of analytes that can be detected using this type of assay. The use of rapid chromatographic techniques for detection of the presence of an analyte in a biological sample has thus progressed beyond the bounds of the clinical laboratory, as assay devices employing these techniques have been found to be especially valuable in xe2x80x9cpoint of carexe2x80x9d situations such as the doctor""s office or home settings.
The typical rapid chromatographic tests utilize either a xe2x80x9csandwichxe2x80x9d assay or a xe2x80x9ccompetitionxe2x80x9d assay to detect the presence of a desired analyte. In the sandwich assay, an analyte is bound, or xe2x80x9csandwiched,xe2x80x9d between an unlabeled first binding partner and a labeled second binding partner. For example, an analyte, such as an antibody to HIV, can be captured by a first binding partner, in this case, an HIV antigen immobilized on a membrane. The antibody-antigen complex can then be detected by a second binding partner having a label, such as another HIV antigen tagged with a colored particle.
In contrast, during the competition assay, the analyte in the sample competes with a labeled analyte, or labeled analogue to the analyte, for a binding partner immobilized on a solid support. A greater concentration of analyte in the sample results in a lower signal in the assay, as the labeled analytes are competed away from the binding partner on the solid support (i.e., the signal produced during a competition assay decreases as the concentration of analyte in the sample increases). Thus, the sandwich assay provides a qualitative assessment with great sensitivity, while the competition assay provides a quantitative measure of analyte concentration.
Regardless of the analyte-detecting method used, the rapid assay devices currently available are often categorized into one of three basic formats: the xe2x80x9cdipstickxe2x80x9d format, the xe2x80x9cflow throughxe2x80x9d format, and the xe2x80x9clateral flowxe2x80x9d format. The xe2x80x9cdipstickxe2x80x9d format (exemplified in U.S. Pat. Nos. 5,275,785, 5,504,013, 5,602,040, 5,622,871 and 5,656,503) typically consists of a strip of porous material having a sample receiving end, a reagent zone and a reaction zone. The sample is wicked along the assay device starting at the sample-receiving end and moving into the reagent zone. The analyte to be detected binds to a reagent incorporated into the reagent zone, preferably a labeled binding partner, to form a complex. Typically, these binding pairs are antibody:antigen complexes, or a receptor:ligand complexes having a label such as a colloidal metal incorporated into the reagent portion of the complex. The labeled binding partner-antigen complex then migrates into the reaction zone, where the complex is captured by another specific binding partner firmly immobilized in the reaction zone. Retention of the labeled complex within the reaction zone thus results in a visible readout.
The xe2x80x9cflow throughxe2x80x9d format (U.S. Pat. No. 4,020,046) also utilizes porous solid phase materials. This assay format usually has a porous membrane that contains an immobilized binding partner positioned above an absorptive layer. Once the sample has been added to the membrane surface, the analyte of interest reacts with the immobilized binding partner to form an analyte-binding partner complex. The complex is visualized by addition of a second binding partner having a label, such as an enzyme, one or more dye particles or various colloidal metals. The absorptive layer acts as a sink for excess assay reagents, and can be used to regulate the flow rate of the reactants to achieve optimal reaction between the analyte and the binding partner. In this format, the sensitivity of the readout can be improved by xe2x80x9cwashingxe2x80x9d the membrane with additional solution to reduce any nonspecific binding of the label, or to remove any other materials which can interfere with the assay readout.
The xe2x80x9clateral flowxe2x80x9d format (see U.S. Pat. Nos. 5,075,078, 5,096,837, 5,354,692 and 5,229,073) utilizes a porous solid phase material and has a linear construction similar to that of the dipstick assay format: a sample application site, a reagent releasing site and a reaction site. However, instead of vertically wicking the samples up the xe2x80x9cdipstick,xe2x80x9d the lateral flow format allows a sample to flow laterally across the porous solid phase material. The sample is applied directly to the application site and the analyte of interest flows laterally to the reagent-releasing site, and forms a complex with a labeled binding partner. The analyte:binding partner complex then migrates into the reaction site where it is captured by a second, immobilized binding partner and detected.
The conventional rapid assays are a popular choice for determining the presence of a given analyte in samples provided at the xe2x80x9cpoint of carexe2x80x9d sites because they are relatively easy to use, do not use specialized equipment or personnel, and produce results in a short amount of time. For example, simple and rapid immunoassay devices for infectious diseases such as AIDS have been available for almost a decade. However, the existing rapid tests are not without their shortcomings. Most importantly, the sensitivity of such devices has often been questioned, due to various limitations with the currently available formats (Giles et al. (1999) Journal of Medical Virology 59:104-109). In addition, there are several practical limitations to the use of these assay devices inherent in the design of the assay format, as exemplified below.
The dipstick format, which was originally designed for urine analysis, uses a relatively large volume of sample for analysis. This is a considerable limitation to use of such a device for analysis of serum or blood samples. In contrast, assay devices based on the flow-through format reduce the volume requirement of samples significantly. However, the flow-through format cannot be employed in a truly self-contained device. In devices based on the flow-through format, the detecting reagent (i.e. the labeled binding partner) is not directly incorporated into the porous solid matrix of device and thus must be provided separately. This leads to additional limitations regarding reagent stability, if the detecting reagents are provided in liquid form, or issues surrounding the proper preparation and handling of the detecting reagent, if provided in a dried form.
The lateral flow format overcomes both the sample volume problem of the dipstick format, as well as the detecting reagent issue of the flow-through format. However, the lateral-flow format does not allow for a washing step, as inherent in the flow-through format. Any interfering species, such as particulate or colored material introduced by the sample solution, or unbound label, can potentially interfere with the readout of the assay device. As a result, the lateral flow format often employs filtration during the assay procedure, e.g., using specially coated filters to remove potential interfering species prior to detection of the analyte. (see, for example, U.S. Pat. Nos. 4,933,092, 5,452,716, and 5,665,238)
A number of clinical conditions are (or could be) monitored using one or more rapid assay devices. For example, Helicobacter pylori has been identified as a pathogen leading to chronic gastritis, peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma (Huang et al. (1998) Gasteroenterology 114:1169-79). The conventional xe2x80x9cgold standardxe2x80x9d tests typically involve invasive endoscopy, followed by histology, culture or rapid urease tests, all of which necessitate a hospital laboratory setting and specially trained medical personnel. On the other hand, the near-patient whole blood or serum/plasma based rapid test devices that have recently become available have not lived up to expectations. There are mixed reports regarding the performances of these kits, particularly in correlation to the ethnic profile of sera being examined. Although some of these rapid test kits perform with approximately 90% sensitivity, often these same kits are compromised by lower performances in specificities, especially when used in different geographical territories. The reverse is also true of kits with high specificities but low sensitivities (see Enroth et al. (1997) J. Cling. Micro. 35:2695-97; Stone et al. (1997) Eur. J. of Gastroenterol. and Hepatology 9:257-260; Hackelsberger et al. (1998) Helicobacter 3:179-183; Leung et al. (1998) J. Clin. Micro. 36:3441-3442). For example, when used to test Asian populations, kits developed using Western serum panels were found to have poorer performance profiles ranging from 63-84% in sensitivity and 82-84% in specificity, considerably lower than those recorded for Western serum panels (Leung, supra). There is an obvious need for an accurate rapid test device for global use, that is both sensitive and specific without compromising one feature for the other.
Tuberculosis (TB) is another example of a clinical condition that would benefit from an improved rapid assay device (for a review of current diagnostic tests, see Andersen et al. (2000) Lancet 356:1099-1104). The re-emerging of this chronic disease is believed to be due largely to the emergence of drug-resistant strains of M. tuberculosis, in concert with a demonstrated increase in risk of infection among human immunodeficiency virus (HIV)-infected populations (for reviews, see Daley et al. (1992) New Engl. J Med. 23;326(4):231-235; Havlir and Barnes (1999) New Engl. J Med. 4;340(5):367-373; Schaaf et al. (1996) Trop Med. Int. Health Oct;1(5):718-722; and Selwyn et al. (1989) New Engl. J Med. 2;320(9):545-550). Acid-fast bacilli (AFB) microscopy employing a Ziehl-Neelson staining protocol is currently the primary diagnostic and monitoring technique, despite the inherent lack of sensitivity and stringent assay requirements (Perkins (2000) Int. J. Tuberc. Lung Dis. 4(12):51-57; Periera et al (2000) J. Clin. Microbiol. 38:2278-2283). On the other hand, the usefulness of currently available serological tests is debated (Freeman et al (1999) J. Clin. Microbiol. 37(6):2111-2112; Rasolofo and Chanteau (1999) J. Clin. Microbiol. 37(12):4201; Desem and Jones (1998) Clin. Diag. Lab. Immunol. 5:531-536). A recent evaluation of seven currently available serological tests revealed that the sensitivities of such tests are lower than previously reported (Pottumarthy et al. (2000) J. Clin. Microbiol. 38(6):2227-31). Furthermore, the sensitivities of standard serological tests are often heavily diminished in tuberculosis patients co-infected with HIV. There is still an unmet need for new rapid assay devices, particularly those that provide a rapid, inexpensive and accurate test for the diagnosis of TB.
The present invention provides novel assay devices, test kits, and methods for detecting the presence of one or more analytes in a sample. The novel approach of the present invention provides optimal control of the assay reactions without requiring specially-developed specific antibodies, large volumes of sample, or complicated arrays of reagents or fluid pathways (for example, as compared to that described in U.S. Pat. Nos. 4,960,691 and 5,607,863). The present invention presents assay devices that are particularly suitable for rapid chromatographic assays using a controlled series of reactions. By controlling the release of the different reagents used in the assay device, the sensitivity of the assay is improved as compared to conventional assays, without compromising the specificity of the assay. The assay devices use a small volume of sample and achieve a much higher titration-end-point activity than conventional lateral flow assays. In addition, the assay devices of the present invention provide better assay sensitivity, without compromising specificity, a highly desirable improvement in the field of rapid chromatographic detection. In addition, the assay can be performed by untrained personnel in a minimum amount of time, and without the need for specialized equipment.
Accordingly, the present invention provides assay devices for use in detecting the presence of an analyte. One embodiment of the assay device of the present invention includes (a) a chromatographic element comprising a sample receiving end, a reagent releasing end, and a reaction zone; (b) an absorbent pad; and (c) a separator positioned between the chromatographic element and the absorbent pad. The separator employed in the present device includes, but is not limited to, a fluid-impermeable barrier, semi-permeable membrane, a material which dissolves over time upon exposure to an aqueous solution, and the like. Using an assay device of this first embodiment, a sample is applied to the sample-receiving end of the chromatographic element and allowed to migrate laterally by capillary action towards the reagent-releasing end. After the sample covers the reaction zone and the analyte within the sample has interacted with at least one first binding partner immobilized within the reaction zone, an aqueous solution is added to the reagent releasing end of the chromatographic element. The separator is removed (or partially removed) from the device, allowing the absorbent pad to come into contact with the chromatographic element. The aqueous solution can be added prior to the removal of the separator, concurrently with the removal, or immediately afterwards. The separator can be removed by pulling the separator entirely from the assay device, or it can be partially removed such that the sample receiving end of the chromatographic element and the absorbent pad come into contact. One or more reagents embedded at the reagent-releasing end, such as a second binding partner labeled with a detectable label such as a naturally colored particle, are released by addition of the aqueous solution and moved toward the reaction zone by the pulling force of the absorber pad. Thus, the device according to this embodiment allows the analyte to form a complex with the first binding partner prior to the reaction between the labeled second binding partner and the bound analyte complex. In addition, the aqueous solution added to the reagent releasing end of the chromatographic element acts not only as a reagent releasing solvent but also as a wash liquid. As a result, a visual readout with a clear background is observed within the reaction zone.
Another embodiment of the assay devices of the present invention includes (a) a chromatographic element comprising a sample receiving end having a releasable first binding partner, a reaction zone having an immobilized second binding partner, and a reagent releasing end having a releasable third binding partner containing a label; (b) an absorbent pad; and (c) a separator positioned between the chromatographic element and the absorbent pad. This embodiment of the assay device is preferred when a capture assay is desired. Using an assay device of this second embodiment, the analyte (for example, an antibody) reacts with at least one first binding partner (such as an antigen or a recombinant protein) impregnated at the sample receiving end of the chromatographic element. The analyte-binding partner complex then migrates to the reaction zone, where this first complex is captured by an immobilized second binding partner (the xe2x80x9ccapturing reagentxe2x80x9d, such as anti-human IgG or anti-human IgM antibodies) to form a second complex. When the aqueous solution is added and the separator is removed, one or more third binding partners labeled with a detectable label, such as a naturally colored particle, are released from the reagent releasing end of the chromatographic element, and allowed to laterally flow to the reaction zone. Detectable labels include moieties which can be detected by visual inspection (e.g., moieties which include or produce colored elements), or with the aid of artificial detection systems, including. e.g., optical systems, spectroscopic systems, radiographic systems, or the like. For simplicity of operation, visually detectable labels are preferred.
The third binding partner can interact with the second complex to form a third complex, which can be detected via the label incorporated in the third binding partner. Optionally, the first binding partner is single antigen or a mixture of antigens, and a generic reagent is used as the third labeled binding partner. For example, the generic reagent optionally is an anti-GST antibody, which will react with all GST-constructed recombinant antigens.
Similarly, a third embodiment of the present invention encompasses the use of two or more reagents interacting at the reagent releasing end of a chromatographic element prior to migration across the reaction zone. In this embodiment of the present invention, the assay devices comprise (a) a chromatographic element comprising a sample receiving end, a reaction zone having an immobilized first binding partner, and a reagent releasing end having two releasable binding partners, at least one of which carries a label; (b) an absorbent pad; and (c) a separator positioned between the chromatographic element and the absorbent pad. Using an assay device of this third embodiment, the first complex is formed at the reaction zone between an analyte and a first binding partner bound to the reaction zone. The second reaction occurs at the reagent releasing end between the second and third binding partners once the aqueous solution has been added, to form a second complex bearing a label. The third reaction takes place in the reaction zone, when the [analyte:binding partner] first complex and the [second binding partner:third binding partner] second complex interact to form a third, labeled complex which can be detected. As in the embodiment described above, the second (embedded) binding partner is optionally a single antigen or a mixture of antigens, while the third (labeled) binding partner acting as the detector is optionally a generic reagent such as an anti-GST antibody.
The previous embodiments of the present invention address changes in the reagents used in the assay, and in the order in which the reactions take place. Yet another embodiment of the present invention involves the composition of the separator component of the assay device. Rather than using a barrier that must be manually removed during the assay, the separator can be composed of a material that will provide a xe2x80x9ctime-controlledxe2x80x9d barrier, such as a semi-permeable membrane or a material that dissolves over time. When the device is in use as according to this embodiment of the present invention, by the time that the sample added to the sample receiving end has migrated laterally and covered the reaction zone, the separator will be dissolved or permeable, and the absorbent pad is readied for operation. An aqueous solution can then be added and the assay completed.
In yet other embodiments of the present invention, methods for detecting an analyte in a sample are provided, as are test kits employing the various embodiments of the assay device. Other permutations of the present invention are also possible, such as the simultaneous detection of multiple analytes using a single sample and a single device. Regardless of the embodiment employed, the assay device of the present invention does not need to include any additional filtration techniques using filters with special coatings, as employed in conventional lateral flow devices. The assay device is versatile and can be used to assess a variety of biological fluids or samples including, but not limited to saliva, serum, whole blood, urine, and solubilized fecal samples. This versatility is achieved by controlling the order in which the reactions occur, and by the additional xe2x80x9cwashingxe2x80x9d of the reactants as provided by passage of the aqueous solution though the chromatographic element and into the absorbent pad.
An additional benefit of the present invention is that the simplicity of the design of the assay device provides a generic platform versatile enough to accommodate the needs and requirements for different product lines. An assay device specific for detection of a particular analyte can be easily adapted to detect a different analyte with minimal modification to the overall design, such as replacing the binding partner immobilized within the reaction zone, but still using a xe2x80x9cgenericxe2x80x9d labeled binding partner for detection purposes. There is not any need for the development of additional specialized reaction reagents for the detection of each desired analyte. This not only reduces the time needed to design and produce new assay devices, but also significantly reduces the costs for product development. Furthermore, since the major components of the assay device are the same, manufacturing parameters can be maintained without major changes. Thus, a production facility for manufacture of a series of products based on the assay device of the present invention utilizes the same equipment and a minimal inventory of raw materials for the manufacture of all of the products, which in turn reduces the cost of operation significantly.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.